Purification and characterization of apolipophorin III from immune hemolymph of Heliothis virescens pupae.

Apolipophorin III (ApoLp-III) from Heliothis virescens pupae was purified by heat-treatment followed by Sephadex G-50 filtration and reverse phase-HPLC. The molecular mass of the purified ApoLp-III was determined as 17965.9+/-5 Da by mass spectrometry. The N-terminal sequence confirmed the protein as ApoLp-III with homology of 56-83% to other insect ApoLp-III molecules. The amino acid spatial arrangement of the predicted alpha-helix 1 of Heliothis ApoLp-III was nearly identical to that of the amphipatic alpha-helix 1 of Manduca sexta ApoLp-III. The absorption spectrum from 240-340 nm of the Heliothis ApoLp-III was the same as the UV spectra of ApoLp-III from Manduca sexta and Galleria mellonella, showing absorption maxima at 280, 268, 264 and 259 nm. These results indicated that the primary structure of ApoLp-III is conserved in lepidopterans. The Heliothis ApoLp-III was not a glycoprotein and showed hemagglutination activity against rabbit red blood cells. This hemagglutination activity was abolished by Tween 80, but not by six different carbohydrates. Hydrophobic interaction of ApoLp-III with red blood cells agreed with structural studies since ApoLp-III binds lipid through hydrophobic interaction after conformational change. Bacterial injection apparently increased the amount of ApoLp-III in immune hemolymph when compared with normal hemolymph, and may indicate that ApoLp-III plays a role in insect immunity.